HGF-mediated S100A11 overexpression enhances proliferation and invasion of gastric cancer - PubMed (original) (raw)

. 2018 Nov 15;10(11):3385-3394.

eCollection 2018.

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HGF-mediated S100A11 overexpression enhances proliferation and invasion of gastric cancer

Sung Ae Koh et al. Am J Transl Res. 2018.

Abstract

S100 proteins are a group of low molecular weight (10-12 kDa) acidic proteins belonging to the largest family of EFhand calciumbinding proteins. S100A11, also known as S100C or calgizzarin, is an important member of the S100 family. S100A11 overexpression has been reported in a number of cancers including papillary thyroid carcinoma, colon, pancreatic, and breast cancer. One other study demonstrated that increased S100A11 expression is correlated with gastric cancer metastasis and poor overall disease prognosis. This study aimed to identify the function of S100A11 associated with cell proliferation and invasion in gastric cancer. We used cell culture, western blotting, reverse transcription-polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and S100A11 knock-down with short hairpin ribonucleic acid (shRNA). First, we confirmed that the S100A11 expression was upregulated by hepatocyte growth factor (HGF). The role of S100A11 was determined via knock down of S100A11. S100A11-shRNA cells showed decreased levels of metalloproteinase-9 (MMP9) and nuclear factor kappa-B (NF-κB). We also examined and confirmed the role of HGF-mediated S100A11 expression. HGF-mediated cell proliferation and in vitro invasion increased, and HGF-mediated apoptosis decreased in S100A11 knockdown cells. We identified the putative binding site of NF-κB in the MMP9 promoter region and confirmed its function via chromatin immunoprecipitation (CHIP) assay. Our results showed that S100A11 is upregulated by HGF through the NF-κB pathway in gastric cancer and plays a role in cell proliferation and invasion in gastric cancer. It may thus be a possible target for gastric cancer therapy.

Keywords: HGF; S100A11; gastric cancer; invasion.

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Conflict of interest statement

None.

Figures

Figure 1

Figure 1

Overexpression of S100A11 level with HGF in NUGC-3 and MKN-28 cells. Cells were serum-starved for 24 h, treated with/without HGF (10 ng/mL) for the indicated times, and harvested. The expression levels of S100A11 RNA and protein were confirmed by RT-PCR (A) and western blot analysis (B). Representative data from three independent experiments are shown. HGF, hepatocyte growth factor; RNA, ribonucleic acid; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 2

Figure 2

Dose effects of HGF on S100A11 expression in NUGC-3 and MKN-28 cells. Cells were serum-starved for 24 h, treated with HGF (0, 10 and 40 ng/ml) for 1 h, and harvested. S100A11 expression levels were confirmed by western blotting. Representative data from three independent experiments are shown. HGF, hepatocyte growth factor.

Figure 3

Figure 3

Overexpression of MMP9 level with HGF in NUGC-3 and MKN-28 cells. Cells were serum-starved for 24 h, treated with HGF (0, 10 and 40 ng/ml) for 1 h, and harvested. The MMP-9 protein levels in culture media were analysed by western blotting (A) and zymography (B). Representative data from three independent experiments are shown. MMP9, metalloproteinase-9.

Figure 4

Figure 4

Effects of LY, PD, SB on the S100A11 and NF-κB expression. The treatment of LY showed the decreased level in both S100A11 and NF-κB expression level in both NUGC-3 and MKN-28 cell lines. The cells (1 × 106/well) were plated overnight in complete medium, starved for 24 h, and then treated with or without LY, PD, SB does dependent for 1 h prior to incubation with or without 10 ng/mL of HGF and harvested. The LCN2 expression was analyzed by Western blotting. This illustrates representative data from three independent experiments. NF-κB, nuclear factor kappa-B; HGF, hepatocyte growth factor.

Figure 5

Figure 5

Down regulation of S100A11 and MMP-9 expression with LY. Cells (1 × 106/well) were plated overnight in complete medium, starved for 24 h, and then treated with or without LY at different doses for 1 h prior to incubation with or without 10 ng/ml of HGF for 48 h, and then harvested. S100A11 expression was analyzed by western blotting. MMP-9 secreted in the media was analyzed by western blotting and zymography. Representative data from three independent experiments are shown. MMP, metalloproteinase; HGF, hepatocyte growth factor.

Figure 6

Figure 6

Effects of NF-κB on S100A11 expression. The expression level of S100A11 was decreased with PDTC treatment. Serum-starved cells were pre-treated with different doses of LY294002 for 45 min, incubated with 10 ng/ml of HGF for 15 min, and harvested. The NF-κB expression levels were confirmed by western blotting (A, B). Serum-starved cells were pretreated with/without PDTC (100 µM) for 45 min, then incubated with 10 ng/mL of HGF for 15 min, and harvested. LCN2 expression levels were confirmed by western blotting (C). Representative data from three independent experiments are shown. NF-κB, nuclear factor kappa-B; HGF, hepatocyte growth factor; PDTC, pyrrolidine dithiocarbamate; LCN2, lipocalin 2.

Figure 7

Figure 7

Down regulation of MMP9, NF-κB protein expression in S100A11 knockdown cells. Control cells and stable S100A11-shRNA cells (1 × 106/well) were plated overnight in complete medium, starved for 24 h, treated with/without 10 ng/mL HGF for 1 h, and harvested. For MMP9 analysis, control cells and stable S100A11-shRNA cells (1 × 106/well) were plated overnight in complete medium, starved for 24 h, treated with/without 10 ng/ml HGF for 48 h, and the spent medium was harvested. The expression levels of MMP9, NF-κB, and S100A11 were analyzed by western blotting. Representative data from three independent experiments are shown. MMP9, metalloproteinase-9; NF-κB, nuclear factor kappa-B; shRNA, short hairpin ribonucleic acid.

Figure 8

Figure 8

Effect S100A11 knockdown on MMP9 promoter activation by HGF. CHIP assay results show the amplification of a fragment of the proximal MMP9 promoter containing the NF-κB binding site but not observed the amplification in S100A11 knockdown cells. Immunoprecipitation was carried out using an anti-NF-κB antibody. Representative data from 3 independent experiments are shown. MMP9, metalloproteinase-9; HGF, hepatocyte growth factor; CHIP, chromatin immunoprecipitation; NF-κB, nuclear factor kappa-B.

Figure 9

Figure 9

Down regulation of S100A11 knockdown on cell proliferation. Control cells (1,000/well) and stable S100A11-shRNA cells were seeded in 96-well plates with DMEM supplemented with 5% FBS and incubated for 24 h. After serum-starvation for 24 h, cells were treated with/without 10 ng/mL HGF for 72 h. Cell proliferation was measured using MTT assays and expressed as a percentage of HGF-untreated control cells. Values are means ± SD of three independent experiments performed in triplicate. shRNA, short hairpin ribonucleic acid; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HGF, hepatocyte growth factor; SD, standard deviation.

Figure 10

Figure 10

Down regulation of S100A11 knockdown on HGF-mediated cell invasion. Stable S100A11-shRNA cells and control cells were treated with/without 10 ng/mL HGF for 48 h. Cell invasion capacity was measured using the standard two chamber invasion assay with Matrigel-coated migration chambers. Values are means ± SD of three independent experiments. HGF, hepatocyte growth factor; shRNA, short hairpin ribonucleic acid; SD, standard deviation.

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